scholarly journals Liver Organoid and T Cell Coculture Models Cytotoxic T Cell Responses against Hepatitis C Virus

2021 ◽  
Author(s):  
Vaishaali Natarajan ◽  
Camille R Simoneau ◽  
Ann Erickson ◽  
Nathan L Meyers ◽  
Jody L Baron ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Nonstructural Protein ◽  
Cytotoxic T Cell ◽  
Effective Vaccine ◽  
Coculture System ◽  
Liver Organoids

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV nonstructural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

scholarly journals Determinants of in vitro expansion of different human virus-specific FoxP3+ regulatory CD8+ T cells in chronic hepatitis C virus infection

2009 ◽  
Vol 90 (7) ◽  
pp. 1692-1701 ◽  
Author(s):  
Eva Billerbeck ◽  
Nobuhiro Nakamoto ◽  
Bianca Seigel ◽  
Hubert E. Blum ◽  
Kyong-Mi Chang ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Cell Populations ◽  
Antigen Specificity ◽  
Specific Stimulation ◽  
Differentiation Status ◽  
Antigen Presenting

It has been shown previously that suppressive virus-specific FoxP3+ regulatory CD8+ T cells can be expanded from human peripheral blood mononuclear cells after in vitro antigen-specific stimulation. This study extended this finding by analysing the mechanisms of virus-specific FoxP3+ regulatory CD8+ T-cell generation during peptide-specific expansion in vitro. It was shown that hepatitis C virus (HCV)-, influenza virus (FLU)-, Epstein–Barr virus (EBV)- and cytomegalovirus (HCMV)-specific FoxP3+ regulatory CD8+ T cells could be expanded differentially from the blood of chronically HCV-infected patients following in vitro peptide-specific stimulation. The different ability of virus-specific CD8+ T-cell populations to express FoxP3 after continuous antigen stimulation in vitro correlated significantly with the ex vivo differentiation status. Indeed, CD27+ CD28+ CD57− HCV-, FLU- and EBV-specific CD8+ T cells displayed a significantly higher ability to give rise to FoxP3+ regulatory CD8+ T cells compared with CD27− CD28− CD57+ HCMV-specific CD8+ T cells. Similar T-cell receptor expression patterns of FoxP3+ versus FoxP3− CD8+ T cells of the same antigen specificity indicated that both cell populations were probably expanded from the same virus-specific CD8+ T-cell precursor. In addition, no specific antigen-presenting cell populations were required for the generation of FoxP3+ CD8+ T cells, as CD8+-selected virus-specific FoxP3+ CD8+ T cells could be expanded by peptide presentation in the absence of antigen-presenting cells. Taken together, these results suggest that the ability to expand FoxP3+ regulatory CD8+ T cells from virus-specific CD8+ T cells differs among distinct virus-specific CD8+ T-cell populations depending on the differentiation status.

scholarly journals Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses

2015 ◽  
Vol 89 (16) ◽  
pp. 8304-8317 ◽  
Author(s):  
Shihong Zhang ◽  
Rakesh K. Bakshi ◽  
Pothakamuri Venkata Suneetha ◽  
Paraskevi Fytili ◽  
Dinler A. Antunes ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
T Cell Responses ◽  
Cross Reactivity ◽  
T Cell Response ◽  
Memory Cells ◽  
Cell Responses

ABSTRACTT cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8+T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show thatin vitroHCV NS3-1073-specific CD8+T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure.Ex vivo, these NS3-1073-specific CD8+T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8+T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8+T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed anin vitroresponse toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine.IMPORTANCEPreventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8+T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.

scholarly journals According to Hepatitis C Virus (HCV) Infection Stage, Interleukin-7 Plus 4-1BB Triggering Alone or Combined with PD-1 Blockade Increases TRAF1low HCV-Specific CD8+ Cell Reactivity

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Elia Moreno-Cubero ◽  
Dolores Subirá ◽  
Eduardo Sanz-de-Villalobos ◽  
Trinidad Parra-Cid ◽  
Antonio Madejón ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Response ◽  
Cell Reactivity ◽  
Interleukin 7 ◽  
Tgf Β1

ABSTRACT Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (pentamer-positive [pentamer+]/CD8+ T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo. Short/midduration PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers. IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+ T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8+ T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

scholarly journals Tetramer Enrichment Reveals the Presence of Phenotypically Diverse Hepatitis C Virus-Specific CD8+T Cells in Chronic Infection

2014 ◽  
Vol 89 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Katja Nitschke ◽  
Tobias Flecken ◽  
Julia Schmidt ◽  
Emma Gostick ◽  
Matthias Marget ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Mhc Class I ◽  
Class I ◽  
Effector Memory ◽  
Cell Populations ◽  
Memory Phenotype

ABSTRACTVirus-specific CD8+T cells are rarely detectableex vivoby conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+T cells in all chronically HCV genotype 1a-infected, HLA-A*02:01-positive patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two-thirds of these enriched HCV-specific CD8+T-cell populations displayed an effector memory phenotype, whereas, surprisingly, one-third displayed a naive-like phenotype despite ongoing viral replication. CD8+T cells with an effector memory phenotype could not expandin vitro, suggesting exhaustion of these cells. Interestingly, some of the naive-like CD8+T cells proliferated vigorously uponin vitropriming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naive-like CD8+T cells from patients with the consensus sequence in the corresponding T-cell epitope did not expandin vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence.IMPORTANCEIn this study, we analyzed CD8+T-cell responses specific for HLA-A*02:01-restricted epitopes in chronically HCV-infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+T-cell populations in all patients. To further characterize these HCV-specific CD8+T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional, and viral sequence analyses. These data revealed different mechanisms for CD8+T-cell failure in HCV infection, including T-cell exhaustion, viral escape, and functional impairment of naive-like HCV-specific CD8+T cells.

scholarly journals De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro

2006 ◽  
Vol 87 (12) ◽  
pp. 3577-3586 ◽  
Author(s):  
Sonya A. MacParland ◽  
Tram N. Q. Pham ◽  
Shashi A. Gujar ◽  
Tomasz I. Michalak
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
T Lymphocytes ◽  
De Novo ◽  
Lymphoid Cells ◽  
Wild Type ◽  
Human T Lymphocytes

While exploring previous findings that ex vivo treatment of lymphoid cells from Hepatitis C virus (HCV)-infected individuals with T cell-stimulating mitogens augments detection of the residing virus, an in vitro HCV replication system was established, in which mitogen-induced T cell-enriched cultures served as HCV targets and the derived T cells multiplied virus during repeated serial passage. HCV replication was ascertained by detecting HCV RNA positive and negative strands, HCV NS5a and E2 proteins, release of HCV virions and nucleocapsids (confirmed by immunoelectron microscopy) and de novo infection of mitogen-induced T cells prepared from healthy donors. Further, affinity-purified normal human T lymphocytes were also susceptible to HCV infection in vitro and HCV replication was detected in pure T cells isolated from a patient with chronic hepatitis C. These results document that T cells can support propagation of HCV both in vivo and in vitro. The infection system established offers a valuable tool for in vitro studies on the entire cycle of HCV replication, virus cytopathogenicity and evaluation of antiviral agents against wild-type HCV in the natural host-cell milieu.

scholarly journals 345 PERSISTENT HEPATITIS C VIRUS (HCV) INFECTION IMPAIRS HCV-SPECIFIC CYTOTOXIC T CELL REACTIVITY THROUGH Mcl-1/Bim IMBALANCE DUE TO CD127 DOWN-REGULATION

2013 ◽  
Vol 58 ◽  
pp. S143-S144
Author(s):  
J.R. Larrubia ◽  
M.U. Lokhande ◽  
S. García-Garzón ◽  
J. Miquel ◽  
A. González-Praetorious ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Hcv Infection ◽  
Cytotoxic T Cell ◽  
Down Regulation ◽  
Cell Reactivity ◽  
T Cell Reactivity

scholarly journals Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

2012 ◽  
Vol 93 (2) ◽  
pp. 247-258 ◽  
Author(s):  
Anna Pasetto ◽  
Lars Frelin ◽  
Anette Brass ◽  
Anila Yasmeen ◽  
Sarene Koh ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Cytotoxic T Lymphocyte ◽  
Hcv Rna ◽  
High Avidity ◽  
Immune Recognition ◽  
Structural Protein ◽  
Human Hepatoma Cells

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

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